U.S. patent number 6,120,493 [Application Number 09/206,635] was granted by the patent office on 2000-09-19 for method for the introduction of therapeutic agents utilizing an electroporation apparatus.
This patent grant is currently assigned to Genetronics, Inc.. Invention is credited to Gunter A. Hofmann.
United States Patent |
6,120,493 |
Hofmann |
September 19, 2000 |
**Please see images for:
( Certificate of Correction ) ** |
Method for the introduction of therapeutic agents utilizing an
electroporation apparatus
Abstract
A method is provided for the introduction of an agent to a
neoplastic cell for electroporating the cell, including contacting
the cell with an electrode template apparatus. The electrode
template apparatus is utilized to apply an electric field to the
cell in order to introduce the agent into the cell. A method is
also provided for the introduction of an agent to a tissue for
electroporating a cell in the tissue, including contacting the
tissue with an electrode template apparatus. The tissue is
contacted with the agent and a pulse of high amplitude electric
signals is applied to the cell utilizing the apparatus, for
electroporation of the cell with the agent.
Inventors: |
Hofmann; Gunter A. (San Diego,
CA) |
Assignee: |
Genetronics, Inc. (San Diego,
DC)
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Family
ID: |
26685908 |
Appl.
No.: |
09/206,635 |
Filed: |
December 7, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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014291 |
Jan 27, 1998 |
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Current U.S.
Class: |
604/506; 604/21;
607/148; 977/915 |
Current CPC
Class: |
A61N
1/325 (20130101); A61N 1/327 (20130101); A61B
2017/00274 (20130101); A61B 2017/3411 (20130101); Y10S
977/915 (20130101); A61B 2018/00547 (20130101) |
Current International
Class: |
A61N
1/32 (20060101); A61B 17/34 (20060101); A61B
17/00 (20060101); A61M 031/00 () |
Field of
Search: |
;604/21,20,506
;607/108,116,143,148 ;435/173.6 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Bockelman; Mark
Attorney, Agent or Firm: Gray Cary Ware & Freidenrich
LLP Haile; Lisa A.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of U.S. patent
application Ser. No. 09/014,291, filed Jan. 27, 1998, herein
incorporated by reference.
Claims
What is claimed is:
1. A method for the introduction of an agent to a neoplastic cell
for damaging the cell, comprising:
(a) contacting said cell with an electrode template apparatus
comprising
a primary support member having opposite surfaces;
a plurality of bores arranged in rows and extending through said
support member and through said opposite surfaces;
a plurality of conductors on said support member separately
connected to at least one of said plurality of bores;
a plurality of needle electrodes mounted in said plurality of bores
so that each conductor is connected to at least one electrode;
means for connecting said conductors to a power supply;
(b) contacting said cell with said agent either prior to,
simultaneously with or after step (a); and
(c) applying an electric field to said cell by utilizing said
electrode template apparatus to apply an electric field to at least
two pairs of said needle electrodes simultaneously, sufficient to
cause electroporation, thereby introducing said agent to said
cell.
2. The method of claim 1, wherein said contacting is in vivo.
3. The method of claim 1, wherein the cell is selected from the
group consisting of a pancreas, a larynx, a pharynx, a lip, a
throat, a lung, a kidney, a muscle, a breast, a colon, a uterus, a
prostate, a thymus, a testis, a skin, and an ovary cell.
4. The method of claim 3, wherein said cell is a prostate tumor
cell.
5. The method of claim 1, wherein said cell is a mammalian
cell.
6. The method of claim 5, wherein said cell is a human cell.
7. The method of claim 1, wherein said plurality of electrodes is
selected from the group consisting of a four needle array of
electrodes and a six needle array of electrodes.
8. The method of claim 1, wherein the electric field is from about
50 V/cm to 1500 V/cm.
9. The method of claim 1 wherein the electric field is applied as
from about 1 to 10 electrical pulses.
10. The method of claim 9, wherein the electrical pulse is from
about 5 .mu.sec to 50 msec in duration.
11. The method of claim 9, wherein the electrical pulse is selected
from the group consisting of a square wave pulse, an exponential
wave pulse, a unipolar oscillating wave form of limited duration,
an a bipolar oscillating wave form of limited duration.
12. The method of claim 11, wherein said electrical pulse is
comprised of a square wave pulse.
13. The method of claim 1, wherein said agent is a selected from
the group consisting of a nucleic acid, a polypeptide, and a
chemotherapeutic agent.
14. The method of claim 13, wherein said chemotherapeutic agent is
selected from the group consisting of Bleomycin, Cystplatin, and
Mitomycin C.
15. The method of claim 1, further comprising applying a cytokine
to said cell.
16. A method for the introduction of an agent to a tissue for
damaging a cell in the tissue, comprising:
(a) contacting said tissue with an electrode template apparatus,
comprising a primary support member having opposite parallel
surfaces;
a plurality of bores arranged in rows and extending through said
support member and through said opposite surfaces;
a plurality of conductors on said support member separately
connected to at least one of said plurality of bores;
a plurality of needle electrodes mounted in said plurality of bores
so that each conductor is connected to at least one electrode, at
least one of said needle electrodes having a tubular configuration
for injection of said agent into said tissue; and
means for connecting said conductors to a power supply; and
(b) contacting said tissue with said agent either prior to,
simultaneously with or after (a); and
(c) applying a pulse of high amplitude electric signals to at least
two pairs of electrodes simultaneously, sufficient to cause
electroporation of said cell with said agent.
17. The method of claim 16, wherein said contacting is in vivo.
18. The method of claim 16, wherein said cell is a neoplastic
cell.
19. The method of claim 16, wherein the tissue is a mammalian
tissue.
20. The method of claim 19, wherein the tissue is a human
tissue.
21. The method of claim 16, wherein said tissue is selected from
the group consisting of pancreas, larynx, pharynx, lip, throat,
lung, kidney, muscle, breast, colon, uterus, prostate, thymus,
testis, skin, and ovary.
22. The method of claim 21, wherein said tissue is a prostate.
23. The method of claim 16, wherein said plurality of needle
electrodes is selected from the group consisting of a four needle
array of electrodes and a six needle array of electrodes.
24. The method of claim 16, wherein said pulse of high amplitude
electric signals is from about 50 V/cm to 1500 V/cm.
25. The method of claim 24, wherein the pulse of high amplitude
electric signals is applied as from about 1 to 10 electrical
pulses.
26. The method of claim 24, wherein the pulse of high amplitude
electric signals is from about 5 .mu.sec to 50 msec in
duration.
27. The method of claim 24, wherein the pulse of high amplitude
electric signals is selected from the group consisting of a square
wave pulse, an exponential wave pulse, a unipolar oscillating wave
form of limited duration, an a bipolar oscillating wave form of
limited duration.
28. The method of claim 27, wherein said electrical pulse is
comprised of a square wave pulse.
29. The method of claim 16, wherein said agent is a selected from
the group consisting of a nucleic acid, a polypeptide and a
chemotherapeutic agent.
30. The method of claim 29, wherein said chemotherapeutic agent is
selected from the group consisting of Bleomycin, Cystplatin, and
Mitomycin C.
Description
FIELD OF THE INVENTION
This invention relates generally to the field of electroporation,
and more specifically to the use of electroporation to introduce
agents to a neoplastic cell to damage the cell.
BACKGROUND OF THE INVENTION
A cell has a natural resistance to the passage of molecules through
its membranes into the cell cytoplasm. Scientists in the 1970's
first discovered "electroporation", where electrical fields are
used to create pores in cells without causing permanent damage to
them. This discovery made possible the insertion of large molecules
directly into cell cytoplasm. Electroporation was further developed
to aid in the insertion of various molecules into cell cytoplasm by
temporarily creating pores in the cells through which the molecules
pass into the cell.
Electroporation has been used to implant materials into many
different types of cells. Such cells, for example, include eggs,
platelets, human cells, red blood cells, mammalian cells, plant
protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, and
sperm. Furthermore, electroporation has been used to implant a
variety of different materials, referred to herein as "implant
materials", "implant molecules", and "implant agents". These
materials have included DNA, genes, and various chemical
agents.
Electroporation has been used in both in vitro and in vivo
procedures to introduce foreign material into living cells. With in
vitro applications, a sample of live cells is first mixed with the
implant agent and placed between electrodes such as parallel
plates. Then, the electrodes apply an electrical field to the
cell/implant mixture.
With in vivo applications of electroporation, electrodes are
provided in various configurations such as, for example, a caliper
that grips the epidermis overlying a region of cells to be treated.
Alternatively, needle-shaped electrodes may be inserted into the
patient, to access more deeply located cells. In either case, after
the implant agent is injected into the treatment region, the
electrodes apply an electrical field to the region. Examples of
systems that perform in vivo electroporation include the Electro
Cell Manipulator ECM 600 product, and the Electro Square Porator
T820, both made by and available from the BTX Division of
Genetronics, Inc.
In the treatment of certain types of cancer with chemotherapeutic
agents it is necessary to use a high enough dose of a drug to kill
the cancer cells without killing an unacceptably high number of
normal cells. If the chemotherapy drug could be inserted directly
inside the cancer cells, this objective could be achieved. Some of
the best anti-cancer drugs, for example, bleomycin, normally cannot
penetrate the membranes of certain cancer cells effectively.
However, electroporation makes it possible to insert the bleomycin
into the cells.
In general, the treatment is carried out by infusing an anticancer
drug directly into the tumor and applying an electric field to the
tumor between one or more pairs of electrodes. The molecules of the
drug are suspended in the interstitial fluid between and in and
around the tumor cells. By electroporating the tumor cells,
molecules of the drug adjacent to many of the cells are forced or
drawn into the cell, subsequently killing the cancerous tumor cell.
"Electrochemotherapy" is the therapeutic application of
electroporation to deliver chemotherapeutic agents directly to
tumor cells.
Known electroporation techniques (both in vitro and in vivo)
function by applying a brief high voltage pulse to electrodes
positioned around the treatment region. The electric field
generated between the electrodes causes the cell membranes to
temporarily become porous, whereupon molecules of the implant agent
enter the cells. In known electroporation applications, this
electric field comprises a single square wave pulse on the order of
1000 V/cm, of about 100 .mu.s duration. Such a pulse may be
generated, for example, in known applications of the Electro Square
Porator T820, made by the BTX Division of Genetronics, Inc. Needle
electrodes have been found to be very useful in the application of
electroporation to many organs of the body and to tumors in the
body.
An electric field may actually damage the electroporated cells in
some cases. For example, an excessive electric field may damage the
cells by creating permanent pores in the cell walls. In extreme
cases, the electric field may completely destroy the cell. It is
desirable that improved electroporation methods and apparatus with
selectable needle electrode arrays be available.
SUMMARY OF THE INVENTION
The invention provides a therapeutic method utilizing an
electroporation apparatus for the treatment of cells, particularly
a neoplastic cell, in order to damage the cell.
A method is provided for the introduction of an agent to a
neoplastic cell for damaging the cell, including contacting the
cell with an electrode template apparatus. The electrode template
apparatus includes a primary support member having opposite
surfaces, a plurality of bores extending through the support member
and through the opposite surfaces, a plurality of conductors on the
support member separately connected to at least one of the
plurality of bores, a plurality of electrodes selectively
insertable in the plurality of bores so that each conductor is
connected to at least one electrode, and a means for connecting the
conductors to a power supply. The electrode template apparatus is
utilized to apply a high voltage electric field to the cell in
order to introduce the agent into the cell.
A method is provided for the introduction of an agent to a tissue
for damaging a cell in the tissue, including contacting the tissue
with an electrode template apparatus. The apparatus includes a
primary support member having opposite parallel surfaces, a
plurality of bores arranged in a rectangular array and extending
through the support member and through the opposite surfaces, a
plurality of conductors on the support member separately connected
to at least one of the plurality of bores, a plurality of needle
electrodes mounted in the plurality of bores so that each conductor
is connected to at least one electrode, wherein at least one of the
needle electrodes has a tubular configuration for injection of the
agent into the tissue; and a means for connecting the conductors to
a power supply. The tissue is contacted with the agent; and a pulse
of high amplitude electric signals is applied to the cell, for
electroporation of the cell with the agent.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view illustrating a system employing an
exemplary embodiment of the present invention.
FIG. 2 is a side elevation view showing the embodiment of FIG. 1 in
use.
FIG. 2A is an enlarged partial side elevation view illustrating
details of one embodiment of a needle electrode tip.
FIG. 3 is a first layer or PC board of the connector of FIG. 1.
FIG. 4 is a view like FIG. 3 of a second layer of the
connector.
FIG. 5 is a view like FIG. 3 of a third layer of the connector.
FIG. 6 is a view like FIG. 3 of a fourth layer of the
connector.
FIG. 7 is a view like FIG. 3 of a fifth layer of the connector.
FIG. 8 is a view like FIG. 3 of a sixth layer of the connector.
FIG. 9 is a view like FIG. 3 of a seventh layer of the
connector.
FIG. 10 is a perspective view illustrating the positioning of the
layers of FIGS. 3-9 with needles shown in position.
FIG. 11 is a partial sectional view taken along a row of
connectors.
FIG. 12 is a partial sectional view taken across three lines of
conductors of the unit.
FIG. 13 is a schematic illustration of needle electrode arrays.
FIG. 13A is a schematic illustration of a needle electrode array of
FIG. 1 with an alternate electrode connection mode.
FIG. 13B is a schematic illustration of a an alternate needle
electrode array with an alternate electrode connection mode.
FIG. 14 a plan view of a PC board showing circuit connections for
the layout of FIG. 13.
FIG. 15 is a view like FIG. 14 of a second series of connections
for the layout of FIG. 13.
FIG. 16 is a schematic illustration of an alternate embodiment of
an electrode array.
FIG. 17 is a schematic illustration of another embodiment of an
electrode array.
FIG. 18 is a schematic illustration of a further embodiment of an
electrode array.
FIG. 19 is a schematic illustration of a still further embodiment
of an electrode array.
FIG. 20 is a schematic illustration of a system including a pulse
generator and switching circuit connected to an electrode
array.
FIG. 21 is a side elevation view illustrating another embodiment of
the invention showing the needle electrodes mounted in a holder
with the electrodes in the retracted position.
FIG. 22 is a view like FIG. 16 showing the needle electrodes in the
extended position.
FIG. 23 is an enlarged view showing details of the holder of FIG.
21.
FIG. 24 is a side elevation view in section illustrating an
embodiment of the invention like FIG. 21 adapted for a catheter
showing the needle electrodes in the retracted position.
FIG. 25 is a view like FIG. 24 showing the needle electrodes in the
extended position.
FIG. 26 is a perspective view of a catheter embodying the electrode
array of FIG. 24.
FIG. 27 is a side elevation view in section illustrating another
embodiment of the invention in use.
FIG. 28 is a graph of the percentage of PC-3 cells surviving
treatment as compared to the voltage (v) applied in vitro.
FIG. 29 is a graph of the percentage of PC-3 cells surviving
treatment as compared to the bleomycin concentration applied in
vitro. Results are shown for cells treated with bleomycin alone
(.circle-solid.) and for cell treated with bleomycin and
electroporation (.smallcircle.).
FIG. 30 is a graph of tumor volume of human prostate tumor (PC-3)
cells in nude mice. Results are shown for no treatment
(.circle-solid.), treatment with bleomycin alone (.smallcircle.),
and treatment with bleomycin and electroporation
(.tangle-soliddn.).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
It must be noted that as used herein and in the appended claims,
the singular forms "a", "and", and "the" include plural referents
unless the context clearly dictates otherwise. Thus, for example,
reference to "a cell" includes at least one and including a
plurality of such cells and reference to "the needle" includes
reference to one or more needles and equivalents thereof known to
those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
All publications mentioned herein are incorporated herein by
reference in full for the purpose of describing and disclosing the
cells, therapeutic agents, and methodologies which are described in
the publications which might be used in connection with the
presently described invention. The publications discussed above and
throughout the text are provided solely for their disclosure prior
to the filing date of the present application.
The invention provides a method of using an apparatus for the
therapeutic application of electroporation. The method includes
infusion of a chemotherapeutic agent or molecule and
electroporation of the agent or molecule into a tumor. The agent is
injected into tissue, and at least one voltage pulse is applied
between needle electrodes disposed in tissue, wherein the needles
function as the electrodes, thereby generating electric fields in
cells of the tissue. The needle electrode assemblies described
below enable the in vivo positioning of electrodes in or adjacent
to subsurface tumors or other tissue. Such therapeutic treatment is
called electroporation therapy (EPT), a form of
electrochemotherapy. While the focus of the description below is
EPT, the invention may be applied to other treatments, such as gene
therapy of certain organs of the body.
THERAPEUTIC METHOD
The therapeutic method of the invention includes electrotherapy,
also referred to herein as electroporation therapy (EPT), using the
apparatus of the invention for the delivery of an agent to a cell
or tissue, either in vivo or in vitro. The term "agent" or
"molecule" as used herein refers to drugs (e.g., chemotherapeutic
agents), nucleic acids (e.g., polynucleotides), peptides and
polypeptides, including antibodies. The term polynucleotides
include DNA, cDNA and RNA sequences.
A "chemotherapeutic agent" is an agent having an antitumor or
cytotoxic effect. Such agents can be "exogenous" agents, which are
not normally found in the organism (e.g., chemical compounds and
drugs). Such drugs or agents include bleomycin, neocarcinostatin,
suramin, doxorubicin, taxol, mitomycin C and cisplatin. Other
exogenous chemotherapeutic agents will be known to those of skill
in the art (see for example The Merck Index). Chemotherapeutic
agents can also be "endogenous" agents, which are native to the
organism. Endogenous agents include suitable naturally-occurring
agents, such as biological response modifiers such as cytokines, or
hormones.
Therapeutic peptides or polypeptides may also be included in the
therapeutic method of the invention. For example, immunomodulatory
agents and other biological response modifiers can be administered
for incorporation by a cell. The term "biological response
modifiers" is meant to encompass substances which are involved in
modifying the immune response. Examples of immune response
modifiers include such compounds as cytokines. The term "cytokine"
is used as a generic name for a diverse group of soluble proteins
and peptides which act as humoral regulators at nano- to picomolar
concentrations and which, either under normal or pathological
conditions, modulate the functional activities of individual cells
and tissues. Also included are polynucleotides which encode
metabolic enzymes and proteins, including antiangiogenesis
compounds, e.g., Factor VIII or Factor IX.
In electrochemotherapy, electroporation is used to deliver
chemotherapeutic agents directly into tumor cells.
"Electroporation" refers to increased permeability of a cell
membrane and/or a portion of cells of a targeted tissue (or
population of cells) to an agent, when the increased permeability
occurs as a result of an application of voltage across a cell. It
is believed that electroporation facilitates entry of a
chemotherapeutic agent such as bleomycin or other drugs into the
tumor cell by creating pores in the cell membrane. Treatment is
carried out by administering an anticancer drug directly into the
tumor and applying an electric field to the tumor between a pair of
electrodes. Without being bound by theory, the molecules of the
drug are suspended in the interstitial fluid between and in and
around the tumor cells. By electroporating the tumor cells,
molecules of the drug adjacent to many of the cells are forced or
drawn into the cell, subsequently killing the cancerous tumor
cell.
Any cell in vivo can be treated by the method of the invention. The
method of the invention is useful in treating cell proliferative
disorders of the various organ systems of the body. The method of
the invention for the treatment of cells, including but not limited
to the cells of the prostate, pancreas, larynx, pharynx, lip,
throat, lung, kidney, muscle, breast, colon, uterus, thymus,
testis, skin, and ovary. The cells may be cells from any mammal,
including mice, rats, rabbits, dogs, cats, pigs, cows, sheep, and
humans. In a preferred embodiment, the cells are human cells.
The term "neoplasia" refers to a disease of inappropriate cell
proliferation. This derangement is most evident clinically when
tumor tissue bulk compromises the function of vital organs. The
term "cell proliferative disorder" denotes malignant as well as
non-malignant cell populations which often appear to differ from
the surrounding tissue both morphologically and genotypically.
Malignant cells (i.e., tumors or cancer) develop as a result of a
multi-step process. Concepts describing normal tissue growth are
applicable to malignant tissue because normal and malignant tissues
can share similar growth characteristics, both at the level of the
single cell and at the level of the tissue. Tumors are as much a
disease of disordered tissue growth regulation as of disordered
cellular growth regulation. The growth characteristics of tumors
are such that new cell production exceeds cell death; a neoplastic
event tends to produce an increase in the proportion of stem cells
undergoing self-renewal and a corresponding decrease in the
proportion progressing to maturation (McCulloch, E. A., et al.,
"The contribution of blast cell properties to outcome variation in
acute myeloblastic leukemia (AML), Blood 59:601-608, 1982). In one
embodiment, the cells treated by the method of the invention are
neoplastic cells. Thus, the electroporation methods of the
invention can be used to treat cell proliferative disorders.
A number of experiments have been conducted to test therapeutic
application of electroporation for cell proliferative disorders in
a process termed electrochemotherapy. This treatment is carried out
by infusing an anticancer drug directly into the tumor and applying
an electric field to the tumor between a pair of electrodes. The
field strength must be adjusted reasonably accurately so that
electroporation of the cells of the tumor occurs without damaging
significant numbers of normal or healthy cells. This can be carried
out with external tumors by applying the electrodes to opposite
sides of the tumor so that the electric field is between the
electrodes. The distance between the electrodes can then be
measured and a suitable voltage according to the formula E=V/d can
then be applied to the electrodes. The electrode apparatus of use
with the methods of the invention has electrodes that can be
inserted into or adjacent to tumors so that predetermined electric
fields can be generated in the tumor tissue for electroporation of
the cells of the tumor. In one embodiment, the electric field
applied by the apparatus is from about 50 V/cm to 1500 V/cm. The
electrical field can be applied as from about 1 to about 10
electrical pulses. In one embodiment, the electrical pulse is
delivered as a pulse lasting from about 5 .mu.sec to 50 msec in
duration. The electrical pulse can be applied as a square wave
pulse, an exponential wave pulse, a unipolar oscillating wave form
of limited duration, or a bipolar oscillating wave form of limited
duration. In one embodiment, the electrical pulse is comprised of a
square wave pulse.
The electrical pulse can be delivered before, at the same time as,
or after, the application of the agent. The chemical composition of
the agent will dictate the most appropriate time to administer the
agent in relation to the administration of the electric pulse. For
example, while not wanting to be bound by a particular theory, it
is believed that a drug having a low isoelectric point (e.g.,
neocarcinostatin, IEP=3.78), would likely be more effective if
administered post-electroporation in order to avoid electrostatic
interaction of the highly charged drug within the field. Further,
such drugs as bleomycin, which have a very negative log P, (P being
the partition coefficient between octanol and water), are very
large in size (MW=1400), and are hydrophilic, thereby associating
closely with the lipid membrane, diffuse very slowly into a tumor
cell and are typically administered prior to or substantially
simultaneous with the electric pulse. Preferably, the molecule is
administered substantially contemporaneously with the
electroporation treatment. The term "substantially
contemporaneously" means that the molecule and the electroporation
treatment are administered reasonably close together with respect
to time. The administration of the molecule or therapeutic agent
and electroporation can occur at any interval, depending upon such
factors, for example, as the nature of the tumor, the condition of
the patient, the size and chemical characteristics of the molecule
and half-life of the molecule.
Electroporation can help minimize the amount of a chemotherapeutic
agent used, these chemicals frequently being harmful to normal
cells. In particular, less of the chemotherapeutic agent can be
introduced into the tumorous area because the electroporation will
enable more of the implant agent to actually enter the cell.
"Administering" an agent in the methods of the invention may be
accomplished by any means known to the skilled artisan.
Administration of an agent in the methods of the invention can be,
for example, parenterally by injection, rapid infusion,
nasopharyngeal absorption, dermal absorption, and orally. In the
case of a tumor, for example, a chemotherapeutic or other agent can
be administered locally, systemically or directly injected into the
tumor. In one embodiment, when a drug is administered directly into
the tumor the drug is injected in a "fanning" manner. The term
"fanning" refers to administering the drug by changing the
direction of the needle as the drug is being injected or by
multiple injections in multiple directions like opening up of a
hand fan, rather than as a bolus, in order to provide a greater
distribution of drug throughout the tumor. It is desirable to
adjust the volume of the drug-containing solution to ensure
adequate administration to the a tumor, in order to insure adequate
distribution of the drug throughout the tumor. For example, a
typical injection may based on the size, volume, or weight of the
tissue being treated. In one specific, non-limiting example using
dogs described herein (see EXAMPLES), 0.25 ml/cm.sup.3 of
drug-containing solution is injected into the treated tissue. Thus,
the volume of drug containing solution is adjusted based on the
size of the treated tissue. In the human tissues, the volume would
similarly be adjusted to ensure adequate perfusion of the tumor. In
one embodiment, injection is done very slowly all around the base
of a tumor and by fanning in a human subject.
Preparations for parenteral administration include sterile or
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Besides the inert diluents, such
compositions can also include adjuvants, wetting agents,
emulsifying and suspending agents. Further, vasoconstrictor agents
can be used to keep the therapeutic agent localized prior to
pulsing.
ELECTROPORATION APPARATUS
Referring to FIG. 1 of the drawing, an electroporation system 10
embodying an exemplary embodiment useful in the methods of the
present invention is illustrated. The system comprises a pulse
generator 12 for generating high voltage pulses and is preferably
of the type sold under the mark "MedPulser" by Genetronics, Inc.
The pulse generator is preferably of the type disclosed in
application Ser. No. 08/905,240, entitled, "Method of Treatment
Using Electroporation Mediated Delivery of Drugs and Genes", filed
Aug. 1, 1997 (herein incorporated by reference), wherein a user
defined pulse may be selected and various parameters can be
programmed. This enables pre-selectable pulsing schemes suitable
for the particular applications.
The pulsing unit has the usual control panel with a power selector
switch 14 and may also have other controls such as a remote
activation means 16. The panel would also have various indicators
to indicate to the operator various conditions and parameters, such
as a digital readout 18 for therapy set-point. A conductor cable 20
connects the pulse generator to a connector and template 22 for a
plurality of electrodes. The electrode connector and template 22
serves to connect selected electrodes to selected conductors, which
in turn connect the electrodes to the pulse generator. The template
also aids in establishing a pre-determined array or multiple arrays
of electrodes.
A precise and controlled voltage must be applied to the tissue in
order to provide the optimum electroporation or poration of the
cells. Therefore, it is essential that the spacing of the
electrodes be known so that the optimum voltage may be applied
between the selected electrodes. The voltage must be applied in
accordance with the spacing between the electrodes in order to
apply the optimum voltage to the cells. The connector template 22
provides a means of selectively positioning any number of
electrodes in a pre-determined array with pre-determined
spacing.
The illustrated system was initially designed for using needle
electrodes to apply electroporation therapy to prostate cancer.
However, it will be appreciated that this system may be utilized
for any number of external and internal tumors or organs of the
body that can be reached from a body or other surface. For example,
this system will enable the treatment of prostate tumors, breast
tumors, local tumors, pancreatic tumors, liver tumors, or any other
organ within the body that is accessible by needle electrodes or
any other manner including open surgery. While the discussion
herein has been primarily for the insertion of drugs into cells
within tumors, or the like, it will also be appreciated that it can
be used for the insertion of DNA or other genetic materials into
cells within an organ or any selected tissue in the body for
altering or generating a genetic response within an organ in the
body, or within cells in that organ or tissue.
The applicant has found through experimentation that pulsing
between
opposed sets of multiple electrodes such as at least sets of pairs
of electrodes in a multiple electrode array, preferably set up in
parallel, rectangular or square patterns, provides improved results
over that of pulsing between a pair of electrodes. Disclosed, for
example, in application Ser. No. 08/467,566 entitled,
"Electroporation Mediated Delivery of Drugs and Genes" is an array
of needles wherein a plurality of pairs of needles define an area
and may be pulsed during the therapeutic treatment. In that
application, which is incorporated herein by reference as though
fully set forth, needles were disposed in a circular array, but
have connectors and switching apparatus enabling a pulsing between
opposing pairs of needle electrodes.
The connector template of the present invention is designed to
provide a system for accurately establishing a pre-selected array
of needle electrodes, with a pre-determined spacing between the
multiple electrodes, positioned within a tissue where
electroporation is desired. The connector (22) is in the form of a
support body having a plurality of rows of bores through which
needle electrodes may be selectively inserted to define a selected
array and connected via the through holes by conductors to the
pulse generator by a suitable connector such as cable. In the
illustrated embodiment, seven rows of seven bores are provided with
the bores and rows spaced an equal distance apart. The spacing
between the rows may be selected for the particular application,
but an exemplary preferred spacing is on the order of about 0.65
cm. With this arrangement, each needle electrode can be spaced a
distance of 0.65 cm from an adjacent electrode.
The electrodes are positioned in the grid in a selected manner to
cover the desired areas of the tissue and the connections to the
electrodes, such that the needles may be selectively distributed
throughout the area of a tumor such that each square (bound by four
needles or two pairs) within the tumor can be subjected to four
pulses of alternate polarity rotating 90.degree. between pulses.
The switching may be done by electronic means square after square
at a high frequency so that the total treatment time is on the
order of a few seconds. With such an array, high voltages may be
applied to the cells between the electrodes without subjecting
other areas of tissue to uncomfortable voltage or current
levels.
As shown in FIGS. 1 and 2, the exemplary connector template is a
box-like support structure having a front face 24 and a back face
(not shown). A first row of through bores 26, 28, 30, 32, 34, 36,
and 38 are connected on the upper surface by means of conductors
40, 42, 44, 46, 48, 50, and 52 to a side edge of the support
housing where they are connected by suitable means, either directly
or by a plug and socket structure to the cable 20.
Second and subsequent rows of the through holes (not numbered) will
be connected by conductors on the different levels of the laminate
making up the connector structure as will be subsequently
described. This enables a closer spacing of the electrodes. An
exemplary group of needles 58, 60, 62, 64, 66 and 68 are shown in
some of the bores. Certain of the electrodes 60, 64 and 68 are
hollow and have a suitable connector at the outer end to enable the
infusion of drugs or genes. These needles also have one or more
suitable outlets such as an open end or one or more ports at or
near the inserted end. For example, the hollow needle electrodes
are shown to have outlet ports, with the ports of electrode 60
shown to have outlet ports identified by reference numerals 70 and
72.
Referring specifically to FIG. 2, the illustrated connector
template is shown in use in treatment of a prostate cancer or the
like. In this instance, the connector 22 is shown mounted on an
elongated support rod 54 of an ultra-sound probe 56 which is shown
inserted into the rectum of a patient. The sound probe is used to
visualize the prostate and the location of the electrodes in the
prostate. The template is then in a position such that a plurality
of the needle electrodes 58, 60 and 62 are inserted through three
of the horizontal through bores, as illustrated, and into the
prostate of the patient. In this instance, two of the needle
electrodes, 58 and 62, are illustrated as being solid needle
electrodes and a center electrode 60 is shown to be hollow to
enable the injection of molecules, such as a drug or a therapeutic
agent or other material. A second group of needle electrodes 64, 66
and 68 are below the aforementioned electrodes and extend through
the through bores of the connector template and into the prostate
of the patient. In this instance, two of these needles 64 and 68
are hollow to enable the injection of a therapeutic or other agent
into the prostate of the patient. These may be left in place
following the injection of the therapeutic agent and serve as the
electrodes for the application of the electrical pulses to the
tissue of the prostate or cancer cells within the prostate. In one
embodiment of the invention, the needle electrodes are partially
insulated along an intermediate portion of the shaft so that only
that portion in the selected tissue and in the template are
conductive. This positions the conductive path through the selected
tissue to be treated and isolates overlying tissue from the
electrical pulses.
As will be apparent from the foregoing illustration and
description, sufficient needle electrodes may be positioned through
the connector template in substantially any desired array to cover
the necessary area of tissue to which electroporation is to be
applied. The needle electrodes may be constructed of any suitable
electrically conductive materials. By way of example but not
limitation such materials may include platinum, silver, gold,
stainless steel and or alloy of these and/or other materials. In
certain applications the tissue to be treated lies beneath healthy
tissue, the electrodes may preferably be insulated along a portion
of the length to isolate the overlying tissue from the pulses. The
needle electrodes may also take any suitable form and have any
suitable length for the particular application. For example, in an
application wherein insertion into or through hard material such as
bone is necessary, the needle may be formed with a suitable
drilling point such as illustrated in FIG. 2A. Referring to FIG.
2A, a needle electrode 69 is shown formed with a spade type
drilling point 69a for drilling through bone and other hard tissue.
The point may be formed as a twist drill or in any other suitable
drill configuration. The drill point electrode may be rotated by
any suitable power means such as a hand drill or a small hand held
drill motor.
Referring now to FIGS. 3-9, there is illustrated a plurality of
printed circuit boards which are stacked together to make up the
combined template connector 22. A PC board 24, as shown in FIG. 3,
forms the face 24 of the connector template unit. This board, as in
each of the boards, has a dimension of about 5 cm.sup.2. Due to the
small space available for the through holes which include the
connectors for the respective electrodes, separate circuits for
several of the through holes such as each row of the through holes
are put on separate PC boards. Thus, as illustrated in FIGS. 3-9,
separate connectors and conductors for each row of the needle
electrodes that will be inserted in a through hole are formed on
the surface of a separate PC board. These are then stacked in an
array, as illustrated, for example, in FIG. 10. It will be
appreciated that the connections for the respective holes in the PC
boards can be made in any number of arrangements, such as a
vertical or horizontal array.
Referring now to FIG. 4, it will be seen that a PC board 74, which
will be disposed directly below the PC board 24, has a row of
enlarged holes 76-88 which are designed to receive the lower end of
connectors on the board above, as illustrated in FIG. 11. In
addition, this PC board has a row of connectors 90-102 which forms
the second row of connecting holes for the needle electrodes of the
assembly. These connecting sockets are each connected as in the
previous embodiment to separate conductors extending along the
surface of the PC board to an edge of the board where they will be
connected to the cable 2). Each connector is separately connected
through its own conductor into the circuit to the pulse generator
where it can be connected in any desirable manner to the generator.
For example, each needle can be paired with each adjacent needle in
either like polarity or opposed polarity. Thus, the needles can be
pulsed in pairs (i.e., two needles of opposed polarity), in
multiple pairs (i.e., pair against pair), or in opposed rows (i.e.
row against row with odd, even or different numbers of electrodes
in opposition).
Referring now to FIG. 11, a sectional view of a portion of the
connector assembly is illustrated in section. It shows a plurality
of the circuit boards mounted in a frame 114 which supports them in
a slightly spaced relationship, as shown. As illustrated, the
sockets, such as socket 26, for example, comprises a generally
tubular metal shell 116 formed to have an opening 118 at the lower
end, and an opening 120 at the upper or inlet end. The shell is
formed and crimped around spring contacts 122, which is constricted
or bend inward at the center for sliding contact or engagement with
a needle. The socket assembly is of a length to extend through
bores in the upper PC board 24 through bore 76 in the underlying PC
board 74. The socket assemblies are in conductive contact with the
printed circuit conductors on the face of the respective board.
Referring to FIG. 12, the staggered arrangement of the conductors
on the PC boards is illustrated. As illustrated, the second row of
conductors or sockets are formed in the PC board 74, which is
disposed below the PC board 24. The next lower PC board 104 has a
row of conductive sockets, including socket 124 with conductors
running along the surface thereof, as previously described. The
next row of conductive sockets is on the next lower PC board 106,
including a socket 126. Thus, the connectors to the respective
electrodes are disposed on different layers within the array of
circuit boards. This enables the formation of a combined connector
template having very close spacing between the respective
conductors, and thereby enable the provision of a high density
array, as illustrated.
The above described apparatus of the present invention is shown in
use as a prostate cancer electroporation therapy system in FIG. 2
in the illustration. The template is positioned with the plurality
of needles inserted into a prostate 130, as illustrated. In
exemplary embodiment the template is mounted on a handle or
extension 154 of an ultra-sound probe 156 by means of a clamp 136.
The ultra-sound probe is inserted into the rectum of the patient
and utilized by the physician to visualize the tumor in the
prostate. The physician inserts the ultra-sound probe and then
inserts the needles into the tumor through the template.
Thereafter, chemicals are delivered through a plurality of the
needles, which are hollow, into the tumor in the prostate.
Thereafter, electrical pulses are delivered to the needles, in a
suitable switching scheme, such as described in the above
application, or as will be subsequently described. For example, at
least one pulse is initiated between two opposing pairs of needles,
the pulse is then reversed in polarity, then with 90.degree. change
of the needle connection, two more pulses are applied in a first
and then a second polarity.
The above described template array can involve up to 49 electrodes,
each with a separate connection to the pulse generator. It is
desirable in some instances to minimize the number of electrodes
which need to be switched or addressed by the generator. In
alternate embodiments hereinafter described, the arrangement of the
electrodes are in a number of parallel connection so that several
zones can be switched simultaneously thereby reducing the number of
switching required.
Referring to FIG. 13 A, an array of forty-nine needle electrodes is
illustrated wherein all of the electrodes with the same number are
connected in parallel. Thus, every other electrodes in each
horizontal row is connected in parallel. As can be seen, by
switching all electrodes 1 and 2 against needles 3 and 4, then all
electrodes 1 and 3 against all electrodes 2 and 4 and then
reversing the polarity, only four pulses are needed to cover the
entire tissue area between the first row and the second row. Pulses
can be similarly applied between all adjacent rows of electrodes. A
treatment zone is the area between four electrodes, with the
electrodes pulsed in opposed pairs, i.e., a pair of positive
against a pair of negative. The preferred pulsing scheme is one
pulse between the opposing pairs, second pulse between the same
pair in reversed polarity. The switching then rotates 90 degrees to
pair the electrodes 90 degrees to the first pair and pulse with a
first polarity then with an opposite polarity. This pulsing scheme
would be carried out for each row and an adjacent row throughout
the entire array of electrodes with 28 pulses. The effectiveness of
this opposed pairs approach has been verified.
This electrode arrangement can be carried out by a two-layer
circuit board as illustrated in FIGS. 14 and 15, which requires
only 14 connections to the pulse generator. In the illustrated
lay-out, all of the same numbers are connected to the same
conductor connection in parallel. This entire array can be made up
on a two-layer printed circuit board. The principal of switching
zones in parallel can be varied further with so many needles in
parallel that only four pulses are needed to switch the entire
template of 49 needles. Thus, at least multiple, if not all,
treatment zones would be simultaneously pulsed.
Referring to FIG. 13B, an alternative array of twenty-five needle
electrodes is illustrated wherein all of the needles with the same
number are connected in parallel on the circuit board. Thus,
alternate electrodes in both horizontal and vertical rows are
connected in parallel. All suitable electrodes 1 and 2 are pulsed
against needles 3 and 4 which are connected together in parallel;
second pulse is to the same electrodes with reversed polarity;
third pulse electrodes 1 connected to electrodes (3 and pulsed
against electrodes (2 and 4 connected together; fourth pulse in
reverse polarity to this connection. With this connection and
pulsing scheme, any large template with any number of electrodes
can be pulsed with only four pulses.
This switching scheme and variations thereof can be applied to
arrays of any size and substantially any shape. The electrode
arrangement and switching scheme of FIGS. 13A can be carried out by
a two-layer circuit board as illustrated in FIGS. 14 and 15, which
requires only 14 connections to the pulse generator. As illustrated
in FIGS. 14 and 15, a multi-layer connecting template 138 showing a
conductor 140 connecting four of the needle connecting ports in a
first row in parallel. A second layer which may be internal or on
the reverse side of the same board is shown in FIG. 15 with a
conductor 142 connecting the three remaining of the needle sockets
in the first row in parallel. Thus, with this arrangement, seven
conductors on each layer can connect all sockets on the entire
board in this manner to the pulse generator. The sockets of the
circuit board are provided with spring contacts as previously
described, which allow the needle electrodes to make sliding
contact and to be extended and retracted. This enables them to be
easily applied to a design which allows the needles to be extended
from and retractable into a holder.
The above described circuit board systems enables any number of
different arrays of the needle electrodes, preferably with multiple
needles in multiple parallel rows. The needles in each row may be
the same or different in number and may be in direct opposition or
may be offset. In addition to the various arrays of electrodes, the
electrodes may be pulsed in any number of different selectable
arrays and sequences, not necessarily limited by the physical
array. In its broadest sense, it is preferred that multiple
electrodes of one polarity will be pulsed against multiple
electrodes of the opposite polarity. The multiple electrodes will
be at least pairs and may be even or odd in number or may be the
same number in opposition to the same or different number. Several
exemplary optional arrays of electrodes are illustrated in the
following FIGS. 16-19, each of which may have an advantage in
particular applications.
Referring to FIG. 16, a generally rectangular array with alternate
offset rows of needle electrodes is illustrated and designated
generally by the numeral 200. In this array, horizontal rows, such
as 202 and 204 are parallel with electrodes in row 204 less in
number and offset laterally from electrodes in row 202. It will be
seen that vertical rows with alternate offset rows are also formed.
Each electrode in each of the inner shorter row is equally spaced
from two electrodes in each adjacent row. Pairing multiple
electrodes will result in a non-rectangular area of coverage.
Referring to FIG. 17, an electrode array is illustrated wherein
each row of
electrodes have the same number of electrodes and are laterally
offset in one direction one space, and designated generally be the
numeral 206. The alternate rows could be offset in alternate
directions, rather than in the same direction as illustrated. It
will be seen that multiple electrodes are in each row except the
first and last vertical rows. All horizontal rows, such as 208 and
210 have the same number of electrodes, and all vertical rows have
a different number of electrodes. All vertically inclines rows have
the same number with adjacent rows offset one half space. Pairs
have same number of electrodes and are laterally offset in one
direction one space.
Referring to FIG. 18, a generally rectangular electrode array,
designated generally at 212, is illustrated wherein each outermost
row of electrodes have the end electrodes missing. The outside
rows, such as 214 and 216, have fewer electrodes and are shorter
than adjacent inner rows. However, all rows are vertically and
horizontally aligned, so that electrodes may be pulsed in multiple
pairs and multiple opposed electrodes in adjacent parallel
rows.
Referring to FIG. 19, a double circular array of six electrodes is
illustrated forming a generally hexagonal electrode array, and
designated generally by the numeral 218. This array can comprise a
single or multiple hexagonal, with needles 222, 224, 226, 228 and
230 forming one hexagonal. Each hexagon delineates or encircles a
treatment area, wherein each electrode may be paired with each
adjacent electrode in like polarity and switched in polarity. In
this array needles are preferably paired in like polarity against
pairs of opposite polarity. Thus, each electrode (such as 220) is
paired with adjacent electrode 222 in like polarity and is pulsed
against electrodes 226 and 228 which are paired in opposite
polarity. The system includes switch means for pairing each
electrode with an adjacent electrode in any polarity and switch in
polarity progressively around the circle. This array has the
advantage of more thoroughly electroporating the selected area
tissue, because two pulses from each pair will traverse the area.
Opposing pairs of needles are pulsed in sequence around the array
in alternate polarity.
The pulses to the electrodes may be applied in any suitable manner
with any suitable system such as the system diagrammatically
illustrated in FIG. 20. A pulse generator 232 delivers pules 234
via switching circuit 236 to electrodes 240, 242, 244, 246, 248 and
250. The electrodes may be in any selected array. Following each
pulse control means associated with the generator switches the
switching circuit via a signal 238 causing a switching of the
electrodes in polarity and/or pairing. In a preferred arrangement,
the switching circuit may separately connect each electrode in
either polarity and pair it with each adjacent electrode in like or
opposite polarity.
Referring to FIGS. 21-26, an extendable and retractable assembly is
illustrated and designated generally by the numeral 144. The
assembly comprises an elongated central support member 146 having a
head or nose piece 148. A circuit board 150 having a plurality of
sliding through-sockets into which needles are mounted on the
support member 146 and receive the extending and retracting
needles. A plurality of needles 152 are mounted on a tubular sleeve
154 which is mounted on the central support member 146. As the
sleeve is moved along the support member, it alternately extends
and retracts the needles, as illustrated in FIG. 23. The device is
also preferably provided with an indicator or gauge 156 to provide
an indication of the length of extension of the needles. In
operation, the nose piece 148 is placed against the tissue through
which the needles are to extend and the sleeve 154 extended until
the needle electrodes extend to the desired depth. As in previous
embodiments, one or more of the electrodes may be a hollow needle
for the introduction of genes or drugs. A cable 158 connects the
needle electrodes of the device to a pulse generator.
The extendable and retractable needles can also be advantageously
applied to a catheter. Referring to FIGS. 24-26, a catheter tip
assembly is illustrated and designated generally by the numeral
160. In this embodiment, an elongated flexible catheter member 162
is fitted at a distal end with a template 164) having a plurality
of through-sockets with sliding connectors 166, 168, 170 and 172. A
plurality of solid conductor electrodes 174 are mounted in a
moveable actuator plate 176 for movement along the catheter. A
hollow needle 178 for the infusion of nucleic acids or drugs is
mounted in the moveable support plate 176 and extends through one
of the through-sockets and by way of a lumen 180 to a source of
drugs or genes not shown. As shown in FIG. 20, the needle
electrodes may be extended and retracted from the end of the
catheter.
As shown in FIG. 21, the catheter is an elongated flexible member
having the needles at one end and various connectors and
manipulating means at the other end. The infusion lumen 180 extends
to the proximal end of the catheter for connection to a source of
genes or drugs, as the case may be. A plurality of electrode wires
or conductors 182 extend to and through a electrode wire lumen 184.
These extend from the end of the lumen 184 at the proximal end of
the catheter for connection to a suitable pulse generator. A guide
wire 186 extends from the distal end of the catheter and extends
the length thereof by way of a lumen 188). The lumen 188 is
connected at one end to the moveable support 176 and includes a
disk 190 at the proximal end for use in extending and retracting
the needles from the end of the catheter.
FIG. 27 illustrated an area of tissue of the body of a mammal
designated generally by the numeral 160 wherein a selected area of
tissue such as a tumor or an organ 162 is embodied within tissue
164. A plurality of needle electrodes, only one of which 166 will
be described in detail are selected and inserted through the body
tissue 164 into the selected tissue 162. The electrodes are
provided with an insulating coating along a mid portion 168
thereof. A tip portion 170 is left bare in order to provide
conductive contact with tissue 162. An upper portion 172 is also
left bare in order to provide conductive contact with conductive
strip or contacts in bore 174 in PC board 176. This arrangement
enables the electrical pulses bo be applied completely within the
selected tissue 162 without disturbing tissue 164. This feature can
be embodied into any of the previously discussed embodiments of the
apparatus.
The above described systems may employ any number of different
arrays of the needle electrodes, preferably with multiple needles
in multiple parallel rows. The needles in each row may be the same
or different in number and may be in direct opposition or may be
offset. In addition to the physical array of electrodes, the
electrodes may be pulsed in any number of different selectable
arrays and sequences, not necessarily limited by the physical
array. In its broadest sense, it is preferred that multiple
electrodes of one polarity will be pulsed against multiple
electrodes of the opposite polarity. The multiple electrodes will
be at least pairs and may be even or odd in number or may be the
same number in opposition to the same or different number.
The following examples are intended to illustrate but not limit the
invention. While they are typical of those that might be used,
other procedures known to those skilled in the art may
alternatively be used.
EXAMPLE 1
IN VITRO STUDIES OF ELECTROPORATION THERAPY
PC-3 cells (ATCC CRL-1435, a prostate cancer cell line) were grown
in RPMI-1640 supplemented with 15% fetal calf serum (FCS) and 1%
L-glutamine in 5% CO.sub.2 at 37.degree. C. Cells in the
exponential phase of growth were harvested by trypsinization and
their viability was determined by trypan blue exclusion. Cell were
suspended in culture medium at 2.times.10.sup.5 cells/ml and seeded
in the wells of a 96-well plate at a final concentration of
4.times.10.sup.4 cells per well. Cells were pulsed using
appropriate needle array electrodes connected to a square wave
pulse generator. The needle array was inserted in the well of the
96 well microplate and pulsed using the following parameters:
______________________________________ Voltage: 0-1000 v Pulse
Length: 99 .mu.sec Number of Pulses: 6
______________________________________
A cell survival curve was produced for the different electric
fields. The results are shown in FIG. 30. At six pulses of . . .
600 Volts, with a pulse length of . . . .mu.s in a 0.5 cm needle
array, 75-80% of the cells survived 20 hours following treatment.
Thus, these parameters were selected for the electroporation
therapy studies.
Chemotherapeutic agents (Bleomycin, Cisplatin, and Mitomycin C)
were dissolved and diluted in phosphate buffered saline (PBS) and
added directly to the cell suspensions at final concentrations
ranging from 1.3.times.10.sup.-9 M to 1.times.10.sup.-4 M. Cell
survival in the presence of the chemotherapeutic agents, both with
and without the application of the electric field, was determined
by XTT cell proliferation assay 20 hours after treatment (Roehm, N.
W., Rodgers, G. H., Hatfield, S. M., Glasebrook, A. L., "An
Improved Colorimetric Assay for Cell Proliferation and Viability
Utilizing the Tetrazolium Salt XTT," J. Immuol. Methods, 142:2,
257-265, 1991). The XTT assay is based on a spectrophotometric
assay of the metabolic conversion of tetrazolium salts to formazan;
living cells convert XTT to formazan, which can be measured
spectrophotometrically. A sample survival curve is shown in FIG.
28. Results were expresses as a comparison of the IC.sub.50
(concentration of drug inhibiting 50% of the cells) of each agent
in the presence and absence of electroporation, and are presented
in Table 1.
TABLE 1 ______________________________________ EFFECT OF TREATMENT
OF PC-3 CELLS IN VITRO IC.sub.50 IC.sub.50 cytotoxicity Agent no
electroporation with electroporation enhancement ratio
______________________________________ Bleomycin 1 .times.
10.sup.-5 1 .times. 10.sup.-8 1000 Cisplatin 5 .times. 10.sup.-5 1
.times. 10.sup.-5 5 Mitomycin C 8 .times. 10.sup.-5 6 .times.
10.sup.-5 1.33 ______________________________________
The cytotoxic effects of chemotherapeutic agents on PC-3 cells were
significantly enhanced by combining the agents with
electroporation. The highest cytotoxic enhancement was achieved
using Bleomycin and electroporation, followed by Cisplatin and
Mitomycin C. (# of in vitro samples varied between 6 and 9. No
statistics was done, although in diagram, the standard error is
shown). Thus electroporation enhances cell susceptibility to the
cytotoxic agents Bleomycin and Cisplatin.
EXAMPLE 2
MURINE MODEL SYSTEM
In order to examine the effect of electroporation on the
effectiveness of chemotherapeutic agents in vivo, a nude mouse
model was utilized. For these experiments, 0.1 ml of a matrigel
solution (a serum-free solution consisting of one part matrigel
diluted in four parts RPMI-1640) containing 5.times.10.sup.6 PC-3
cells was implanted on the flank of nude mice. The tumors were
allowed to grow until a tumor volume of 80.+-.20 m.sup.3. The mice
were weighed and randomly divided into six groups as follows:
Group 1: no chemotherapeutic agent, no electroporation
Group 2: 0.5 unit Bleomycin, no electroporation
Group 3: 0.5 units Bleomycin, 4 needle array, 0.65 cm, 942V,
4.times.100 .mu.s pulses
Group 4: 0.5 units Bleomycin, 6 needle array, 1.00 cm, 1130 V,
6.times.100 .mu.s pulses
Group 5: 0.5 units Bleomycin, 6 needle array, 0.50 cm, 559 V,
6.times.100 .mu.s pulses
Group 6: 0.5 units Bleomycin, 4 needle array, 0.87 cm, 1500V,
4.times.100 .mu.s pulses
In those animals which received Bleomycin, the chemotherapeutic
agent (0.5 unit) was dissolved in 0.01 ml saline and injected
intratumorally by "fanning." After 10.+-.1 minutes, a Genetronics
Medpulser.TM. device was used to pulse the tumors with a set of
either 6 or 4-needle array electrodes. All treatments were given as
a single set of pulses.
The animals were monitored daily for morality or any sign of
disease for 67 days (see FIG. 24, where D=drug treatment
(Bleomycin) and e=electroporation). Tumor size was measured and
tumor volume calculated using the formula:
wherein a, b, and c are the length, width, and depth of the tumor
in mm.
Following the monitoring period, the tumors were harvested and
sections were prepared for histological analyses. Animals were
classified as having progressive disease (appearance of new lesions
not previously identified or having an estimated increase of 25% or
more in existent lesion size), a complete response (complete
disappearance of all known disease, a partial response (wherein the
tumor size decrease 50% or more). Deaths were noted to be due to
infighting of mice in the same cage.
TABLE 2 ______________________________________ RESULTS OF TREATMENT
OF PC-3 CELLS IN NUDE MICE Group Number of Animals Results
______________________________________ 1 5 4 (80%) Progressive
disease 1 death 2 6 6 (100%) progressive disease 3 7 5 (52%)
complete response 1 (14%) partial response 1 (14%) death 4 7 5
(52%) complete response 1 (14%) partial response 1 (14%) death 5 6
5 (83%) complete response 1 (17%) partial response 6 8 5 (63%)
complete response 1 (12%) partial response 2 (25%) death
______________________________________
The results indicated that the combination of a chemotherapeutic
agent and electroporation is an effective modality for tumor
treatment. Both the 4 and the 6 needle arrays were shown to be
efficacious.
EXAMPLE 3
EVALUATION OF THE TECHNICAL FEASIBILITY OF INTRAPROSTATIC INJECTION
OF BLEOMYCIN
In order to evaluate the technical feasibility of intraprostatic
injection of Bleomycin, the following study was performed. A male
beagle dog with a prostate size of .gtoreq.2 cm in diameter was
anesthetized, A midline laparotomy was performed, and the bladder
and gut reflected to visualize the prostate gland. Under direct
visual guidance, Bleomycin was injected into each of six sextants
(base, mid, and apex of both the left and right sides) of the
prostate. Four electroporation needles were then inserted
transperineally under visual guidance to administer the
electroporation cycles. No acute local or adverse reactions to the
test compound or
electroporation were noted. Small hematomas were evident at the
injection site, which persisted for the duration of the study.
During the electroporation pulses, muscular contractions were
observed. The ECG was recorded during each of the electroporation
pulse sequences. The first two sequences were conducted with the
needles inserted into the prostate, through the perineum. Four
additional sequences were recorded with the needles inserted
directly into the muscles of the left hindlimb. Each of the pulse
sequences produced stimulation artifacts on the recording of the
ECG. However, it was still apparent from the ECG recordings that
there was no effect on the electrical rhythm of the heart, as the
timing of the QRS complexes appeared not to differ during the train
of the electroporation pulses, and no clinical disturbances of the
cardiac rhythm were observed.
One hour after electroporation, the animal was euthanized using
Beuthanasia cocktail, and the prostate, perineum, and surrounding
tissues were examined for gross lesions, in situ. Gross examination
of the prostate, perineum, and surrounding tissues revealed no
findings except for the hematomas on the prostate surface. The
prostate was then excised and processed for histological
evaluation. The significant tissue findings noted in the prostate
gland included hemorrhage, edema, and necrosis, which were mild in
severity and multifocal in distribution pattern. Necrosis occurred
in the epithelial cells in the glandular portion of the prostate.
No necrosis of the supporting stroma was observed. The study
demonstrated that the treatment protocol can be utilized to induce
necrosis of the prostate.
EXAMPLE 4
CANINE MODEL SYSTEM OF INTRAPROSTATIC BLEOMYCIN AND
ELECTROPORATION
In order to investigate the toxicity and side effects of combined
Bleomycin and electroporation in the prostate, a canine model was
evaluated. Male beagle dogs with a prostate size of .gtoreq.2 cm in
diameter are utilized. The following methods are used:
Group 1A, D-E+ (d=drug, E=electric field, +/-=presence or absence,
respectively)
Under general anesthesia, an open laparotomy is performed to expose
the prostate. Electroporation needles are inserted transperitonealy
into the prostate form the base to the apex. of the prostatic
capsule. These are inserted using the square array templated guides
(0.5 cm base length) and transrectal ultrasound (TRUS) ultrasound.
The needle placement and spacing are confirmed with fluoroscopy.
Saline (0.25 ml/cm3) is injected transperitonealy into the
prostate. The injection is delivered to the base, mid and apex
portions of the prostate lobe using the TRUS guidance.
Succinylcholine was given, prior to electroporation, 1 mg/kg, i.v.
An EP pulse is applied according to the following treatment
parameters:
Experiment #1: EPT cycle (658 V) with a four needle array
(1.times.treatment area). Sacrifice at 48 hours
post-electroporation.
Experiment #2: 3 EPT cycles (658 V) with a four needle array
(1.times.treatment area). Sacrifice at 48 hours
post-electroporation.
Electrode position is monitored by TRUS image before, during, and
after electroporation. EKG is monitored before, during and after
electroporation. The toxicity is monitored by examining urination
(void, hematuria) at 0, 24, and 48 hours post electroporation.
Erection (rectal palpitation) is monitored at 0, 24, and 48 hours
post electroporation. The blood chemistry profile (indicating
kidney and liver function) is monitored at 0, 24, and 48 hours post
electroporation. Both gross pathological exam and histopathological
analyses are performed. Specifically, the prostate, testes,
urethra, lung, rectum, kidney, bladder and caudi equina are
examined.
Group 1B, D-E+
Under general anesthesia, an open laparotomy is performed to expose
the prostate. Electroporation needles are inserted
transperitineally into the prostate from the base to the apex of
the prostatic capsule. These are inserted using the square array
templated guides (0.5 cm base length) and transrectal ultrasound
(TRUS) ultrasound. The needle placement and spacing are confirmed
with fluoroscopy. Saline (0.25 ml/cm3) is injected transperitonealy
into the prostate. The injection is delivered to the base, mid and
apex portions of the prostate lobe using the TRUS guidance.
Succinylcholine was given, prior to electroporation, 1 mg/kg, i.v.
An EP pulse is applied according to the following treatment
parameters:
Experiment #3: 3 EPT cycles (658 V) with a four needle array
(1.times.treatment area). Sacrifice at 28 days
post-electroporation.
Electrode position is monitored by TRUS image before, during, and
after electroporation. EKG is monitored before, during and after
electroporation. The toxicity is monitored by examining urination
(void, hematuria) at days 0, 2, 2, 7, 14, and 28 post
electroporation. Erection (rectal palpitation) is monitored at days
0, 2, 2, 7, 14, and 28 post electroporation. The blood chemistry
profile (indicating kidney and liver function) is monitored at days
0, 2, 2, 7, 14, and 28 post electroporation. Both gross
pathological exam and histopathological analyses are performed.
Specifically, the prostate, testes, urethra, lung, rectum, kidney,
bladder and caudi equina are examined.
Group IIA: D+E+
Under general anesthesia, an open laparotomy is performed to expose
the prostate. Electroporation needles are inserted
transperitineally into the prostate from the base to the apex of
the prostatic capsule. These are inserted using the square array
templated guides (0.5 cm base length) and transrectal ultrasound
(TRUS) ultrasound. The needle placement and spacing are confirmed
with fluoroscopy. Bleomycin (4 U/ml) is injected tranperitonealy
into the prostate at 0.25 ml/cm.sup.3 prostate volume (1 U/cm.sup.3
prostate volume) using TRUS guidance. Succinylcholine was given,
prior to electroporation, 1 mg/kg, i.v. An EP pulse is applied
according to the following treatment parameters:
Experiment #4: EPT cycle (658 V) with a four needle array
(1.times.treatment area). Sacrifice at 48 hours
post-electroporation.
#5: 3 EPT cycles (658 V) with a four needle array
(1.times.treatment area). Sacrifice at 48 hours
post-electroporation.
Drug injection and electrode position are monitored by TRUS image
before, during, and after electroporation. EKG is monitored before,
during and after electroporation. The toxicity is monitored by
examining urination (void, hematuria) at 0, 24, and 48 hours post
electroporation. Erection (rectal palpitation) is monitored at 0,
24, and 48 hours post electroporation. The blood chemistry profile
(indicating kidney and liver function) is monitored at 0, 24, and
48 hours post electroporation. Both gross pathological exam and
histopathological analyses are performed. Specifically, the
prostate, testes, urethra, lung, rectum, kidney, bladder and caudi
equina are examined.
Bleomycin pharmacokinetics are also evaluated. Blood levels are
determined at time 0, end of injection and 10, 20, 30, 60 120
minutes post electroporation. The blood level of Bleomycin is
further determined and 12, 24, 36, and 48 hours post
electroporation.
Group IIB, D+E+
Under general anesthesia, an open laparotomy is performed to expose
the prostate. Electroporation needles are inserted
transperitineally into the prostate from the base to the apex of
the prostatic capsule. These are inserted using the square array
templated guides (0.5 cm base length) and transrectal ultrasound
(TRUS) ultrasound. The needle placement and spacing are confirmed
with fluoroscopy. Bleomycin (4 U/ml) is injected tranperitonealy
into the prostate at 0.25 ml/cm.sup.3 prostate volume (1 U/cm.sup.3
prostate volume) using TRUS guidance. Succinylcholine was given,
prior to electroporation, 1 mg/kg, i.v. An EP pulse is applied
according to the following treatment parameters:
Experiment #6: 3 EPT cycles (658 V) with a four needle array
(1.times.treatment area). Sacrifice at 28 days
post-electroporation.
Drug injection and electrode position is monitored by TRUS image
before, during, and after electroporation. EKG is monitored before,
during and after electroporation. The toxicity is monitored by
examining urination (void, hematuria) at days 0, 2, 2, 7, 14, and
28 post electroporation. Erection (rectal palpitation) is monitored
at days 0, 2, 2, 7, 14, and 28 post electroporation. The blood
chemistry profile (indicating kidney and liver function) is
monitored at days 0, 2, 2, 7, 14, and 28 post electroporation. Both
gross pathological exam and histopathological analyses are
performed. Specifically, the prostate, testes, urethra, lung,
rectum, kidney, bladder and caudi equina are examined.
Bleomycin pharmacokinetics are also evaluated. Blood levels are
determined at time 0, end of injection and 10, 20, 30, 60 120
minutes post electroporation. The blood level of Bleomycin is
further determined and 12, 24, 36, and 48 hours and 7, 14, and 28
days post electroporation.
Group IIIA, D+E-
Under general anesthesia, an open laparotomy is performed to expose
the prostate. Bleomycin (4 U/ml) is injected tranperitonealy into
the base, mid, and apex portions of the prostate lobe at 0.25
ml/cm.sup.3 prostate volume (1 U/cm.sup.3 prostate volume) using
TRUS guidance. Succinylcholine was given, prior to electroporation,
1 mg/kg, i.v. The animal(s) are sacrificed 48 hours after Bleomycin
treatment.
Drug injection is monitored by TRUS image before, during, and after
electroporation. EKG is monitored before, during and after
electroporation. The toxicity is monitored by examining urination
(void, hematuria) at 0, 24, and 48 hours post electroporation.
Erection (rectal palpitation) is monitored at 0, 24, and 48 hours
post electroporation. The blood chemistry profile (indicating
kidney and liver function) is monitored at 0, 24, and 48 hours post
electroporation. Both gross pathological exam and histopathological
analyses are performed. Specifically, the prostate, testes,
urethra, lung, rectum, kidney, bladder and caudi equina are
examined.
Bleomycin pharmacokinetics are also evaluated. Blood levels are
determined at time 0, end of injection and 10, 20, 30, 60 120
minutes post electroporation. The blood level of Bleomycin is
further determined and 12, 24, 36, and 48 hours post
electroporation.
Group IIIB, D+E-
Under general anesthesia, an open laparotomy is performed to expose
the prostate. Bleomycin (4 U/ml) is injected tranperitonealy into
the base, mid and apex portions of the prostate lobe at 0.25
ml/cm.sup.3 prostate volume (1 U/cm.sup.3 prostate volume) using
TRUS guidance. Succinyl choline is then injected [PLEASE PROVIDE
DOSAGE]. The animal(s) is sacrificed after 28 days.
Drug injection is monitored by TRUS image before, during, and after
electroporation. EKG is monitored before, during and after
electroporation. The toxicity is monitored by examining urination
(void, hematuria) at days 0, 2, 2, 7, 14, and 28 post
electroporation. Erection (rectal palpitation) is monitored at days
0, 2, 2, 7, 14, and 28 post electroporation. The blood chemistry
profile (indicating kidney and liver function) is monitored at days
0, 2, 2, 7, 14, and 28 post electroporation. Both gross
pathological exam and histopathological analyses are performed.
Specifically, the prostate, testes, urethra, lung, rectum, kidney,
bladder and caudi equina are examined.
Bleomycin pharmacokinetics are also evaluated. Blood levels are
determined at time 0, end of injection and 10, 20, 30, 60 120
minutes post electroporation. The blood level of Bleomycin is
further determined and 12, 24, 36, and 48 hours and 7, 14, and 28
days post electroporation.
Although the invention has been described with reference to the
presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
* * * * *